Identification of clinically important anaerobic bacteria by an oligonucleotide array

Abstract

Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.

FIG. 1.

Layout of oligonucleotide probes on the array (0.7 by 0.7 cm, 7 by 7 dots). Probe PC (C7) was a positive control, and probe NC was a negative control (tracking dye only). Probe M, a position marker, was an irrelevant probe labeled with digoxigenin at the 5′ end. The corresponding species names and sequences of all probes are listed in Table 2.

FIG. 2.

Hybridization patterns of 28 species of anaerobic bacteria and Veillonella spp. All strains except Anaerococcus prevotii CCUG 44020A and Peptoniphilus asaccharolyticus CCUG 12549 were type strains. The corresponding probes hybridized on the arrays are indicated in Fig. 1, and the corresponding sequences of the hybridized probes are shown in Table 2.